Trisomy 21/Down syndrome (DS) is one of the most important human genetic diseases. It currently affects approximately 350,000 people in the United States and more than 2,000,000 people worldwide. It is the most frequent live-born human aneuploidy. It is the most common genetic cause of congenital heart disease and mental retardation. It is a leading cause of gastrointestinal anomalies and megakaryoblastic leukemia. It causes early-onset Alzheimer-type neurodegeneration in nearly every individual with DS. The prevailing hypothesis is that the DS phenotypes are caused by the dosage imbalances of the specific genes on human chromosome (HSA) 21, which is supported by mouse-based experimental results. Many DS phenotypes, including valvuloseptal defects, megakaryoblastic leukemia, and amyloid plaque neuropathology, are highly specific and rarely observed in other human chromosomal disorders, suggesting that these phenotypes are the consequences of the triplications of specific causative genes on HSA 21. However, the efforts to isolate these genes have not been successful due to lacking of an effective approach. In this application, we propose a new genetic analysis strategy for DS based largely on generating and analyzing mouse mutants carrying nested duplications and deletions in the HSA 21 syntenic regions. The strategy is capable of isolating the causative genes for DS phenotypes and predicted to be efficient for the following reasons: (1) We have established the first group of the mouse models of DS trisomic for all of the HSA 21 syntenic regions on mouse chromosomes 10, 16, and 17 and exhibiting DS phenotypes. (2) We have established the MICER resource which provides the ready-made targeting vectors for efficient chromosome engineering in any regions of the mouse genome. (3) The Sanger Institute has approved our request to complete the public effort in generating the knockout mice for all of the orthologs of the HSA 21 genes and has begun the process of mutating 54 of these genes. A major task of studying any human genetic diseases is genetic analysis of the disorders with the goal of isolating the causative genes for the disease phenotypes because subsequent studies on these genes may unravel the true mechanisms of the disorders which are otherwise unattainable. The aims of this discovery-driven proposal are in-depth characterization of DS-associated congenital cardiovascular malformations in our new mutant mice and genetic dissection of this phenotype to identify the critical genomic region(s) and ultimately the causative gene(s). The attainment of our objectives should unravel the entry points to the mechanistic details leading to congenital cardiovascular malformations in DS and may yield rare insights on cardiac development, which may lead to novel strategies for prevention, diagnosis, and treatment of congenital heart disease in children and adults regardless of their states of ploidy. The genetic analysis strategy refined and new mouse mutants generated through this study will have a lasting impact on DS research and will particularly benefit the efforts to isolate causative genes for other major phenotypes of DS.